The present invention relates to novel methods and compositions which utilize catechins including, but not limited to, epigallocatechin-3-gallate (EGCg), epicatechin (EC), epicatechin gallate (ECG), and epigallocatechin (EGC), which are found in varying levels in tea leaves, in combination with vanilloids including, but not limited to, vanillylamine, the vanilloid head group of capsaicin. The compositions of the invention contain various amounts of the catechins and vanilloids, and optionally, other therapeutic agents. The invention also encompasses the varying modes of administration of the catechins and vanillbids as a dietary or nutritional supplement or as a therapeutic compound. There have been reports of antiviral activities of catechins (Mitscher et al., 1994, Med. Res. Rev. 17: 327-365). Inhibition by EGCg and epicatechin-3-gallate (ECG) of both reverse transcriptase at 10 to 20 ng/ml and RNA polymerase has been reported (Nakano & Ono, 1990, Biochemistry 29: 2841-2845; Mizuno, 1992, Planta Medica 5: 535). The effect appeared to be unrelated to antioxidant action, but it seemed to correlate with competition for the template-primer. However, the concentrations of these two catechins required to produce the antiviral effects were toxic to whole cells in culture.
EGCg also has demonstrated a protective effect against infection of cultured rhesus monkey kidney MA104 cells with rotaviruses and enteroviruses (Mukoyama et al., 1991, Jpn. J. Med. Sci. Biol. 44: 181-186). To be most effective, it was necessary to treat the virus with EGCg prior to infection. Treatment of the cells themselves with EGCg either before or after infection produced lesser effects.
Decreased inhibition of infectivity of influenza A and influenza B viruses in cultured Madin-Darby canine kidney cells by EGCg was observed by Nakayama et al.; it was attributed to inhibition of virus adsorption to the target cells and overall agglutination of virus, both as determined by electron microscopy (Nakayama et al., 1993, Jpn. Antiviral Res. 21: 289).
The basis for the anti HIV activity of EGCg in the present is unknown. However, previous work from our laboratory has identified a tumor- (cancer-) associated growth protein, tNOX, as a target for EGCg, the principal anticancer tea catechin, to help explain the anticancer benefits of tea (Morré et al., 2000, Biochem. Pharmacol. 60: 937-946). One of the catalytic activities of this protein that is blocked by EGCg is that of protein disulfide interchange (PDI) (Morré, 1998, Plasma Membrane Redox Systems and their Role in Biological Stress and Disease, Kluwer Academic Publishers, Dordrecht, N L, pp. 121-156).
Enveloped mammalian viruses generally enter cells via fusion of viral and cellular membranes (Battini et al., 1995, J. Virol. 69: 713-719). Thiol-disulfide interchange reactions occur during the interaction of viruses with cells, and these interactions may be necessary for fusion of viral and cellular membranes (Ryser et al., 1994, Proc. Natl. Acad. Sci. USA 91: 4559-4563; Abell & Brown, 1993, J. Virol. 57: 5496-5501). For example, Sindbis virus-induced fusion of cells was enhanced by exogenous reducing agents and inhibited by thiol alkylating agents (Abell & Brown, 1993, J. Virol. 67: 5496-5501). The explanation offered was based on a model in which virion binding to cells led to reduction of critical disulfide bonds in the Sindbis envelope proteins. The result was suggested to be an increased flexibility required for fusion of viral and cellular membranes.
Infection of lymphoid cells by HIV-1 was inhibited by membrane impermeant sulfhydryl blocking reagents and by inhibitors of cell surface PDI (Ryser et al., 1994, Proc. Natl. Acad. Sci. USA, 91: 4559-5463). Implicit in the findings of Ryser et al. (1994, Proc. Natl. Acad. Sci. USA, 91: 4559-5463) was the interpretation that the PDI-like activity mediates a thiol-disulfide exchange with HIV-1 envelope proteins, triggering changes in conformation required for HIV-1 entry.
Demonstrations of the thiol interchange activity of the NOX proteins included the restoration of activity to reduced, denatured and oxidized (scrambled) yeast RNase through reduction, refolding under non-denaturing conditions and reoxidation to form a correct secondary structure stabilized by internal disulfide bonds. The activity resembled that of a classical PDI of the endoplasmic reticulum (Freedman, 1989, Cell 57: 1069-1072) but was clearly due to an activity of a different protein. The EGCg-responsive protein disulfide-thiol interchange activity was not altered by the presence of antisera to PDI (Morré et al., 1997, Biochim. Biophys. Acta 1325: 117-125). Ineffective in blocking tNOX activity were a mouse monoclonal antibody (SPA-891, StressGen Biotechnologies) to PDI from bovine liver (cross-reactive with PDI from human, monkey, rat, mouse and hamster cell lines) and a peptide antibody directed to the characteristic cys-X-X-cys motif common to most, if not all, members of the PDI family of proteins (Chivers et al., 1996, EMBO J. 15: 2659-2667; Bardwell and Beckwith, 1993, Cell 74: 769-771). The EGCg-inhibited NADH oxidation site of the cell surface-located drug-inhibited protein disulfide-thiol interchange protein is located at the external cell surface (Morré et al., 1998, Life Sci. 62: 213-219) and is inhibited by other drugs such as quassinoids (Morré et al., 1994, FEBS Lett. 346: 199-202), brefeldin A (Paulik et al., 1999, Biochem. Pharmacol. 58: 1781-1790) and quassinoid conjugates (Morré, 1995, Biochim. Biophys. Acta 1240: 201-208) all with documented activity in blocking or slowing HIV infection.
Vanilloids are the active ingredients found in Capsicum species, cayenne pepper, black pepper, paprika, cinnamon, clove, mace, mustard, ginger, turmeric, papaya seed and the cactus-like plant Euphorbia resinifera. Vanilloid compounds have been generally disclosed to have analgesic, anti-irritant and anti-inflammatory activities, although certain capsaicinoids are irritants. It is believed that vanilloids mediate their biological effects through vanilloid receptors (reviewed by Caterina & Julius, 2001, Annu. Rev. Neurosci. 24:487-517). C. annuum varieties include the guajillo, cayenne, bell, poblano, serrano, jalapeno, and New Mexican/Anaheim peppers, among others. Representative Capscium frutescens include the Tabasco pepper and the African bird pepper. The especially pungent peppers can be treated with amidase and optionally also with food grade hydrolases, prior to formulation with the tea extracts. Such treatment reduces the apparent “heat” of the pepper preparation and reduces the potential discomfort after consumption.
Capsaicinoids are found in extracts of the fruit (peppers) of the Capsicum species, with high amounts being found in the pungent chili peppers. The capsaicinoids represent a group of natural products that are vanillylamides of monocarboxylic acids of varying chain lengths from C-8 to C-11 with varying degrees of unsaturation.
A unique family of plasma membrane NADH oxidases (NOX), hydroquinone oxidase and protein disulfide-thiol interchange activities (ECTO-NOX-Proteins) that is responsive to hormone and growth factors has been identified (Brightman et al., 1992, Biochim. Biophys. Acta 1105:109-117; Morré, 1994, J. Bioenerg. Biomemb. 26:421-433; and Morré, 1998, Plasma Membrane Redox Systems and their Role in Biological Stress and Disease, Kluwer Academic Publishers, Dordrecht, N L, pp. 121-156). Further, a hormone-insensitive and drug-responsive form of NOX designated tNOX which is specific to cancer cells has been reported (Bruno et al., 1992, Biochem. J. 284:625-628; Morré and Morré, 1995, Protoplasma 184:188-195; Morré et al., 1995, Proc. Natl. Acad. Sci. U.S.A. 92:1831-1835; Morré et al., 1995, Biochim. Biophys. Acta 1240:11-17; Morré et al., 1996, Eur. J. Can. 32A:1995-2003; and Morré et al., 1997, J. Biomemb. Bioenerg. 29:269-280; U.S. Pat. Nos. 5,569,673 and 5,605,810).
Because the ECTO-NOX proteins are located at the external plasma membrane surface and are not transmembrane, primary functional roles as NADH oxidases is not considered likely (Morré, 1994, J. Bioenerg. Biomemb. 26:421-433; DeHahn et al., 1997, Biochim. Biophys. Acta 1328:99-108; and Morré, 1998, Plasma Membrane Redox Systems and their Role in Biological Stress and Disease, Kluwer Academic Publishers, Dordrecht, N L, pp. 121-156). The oxidation of NADH provides for a convenient method to assay the activity. However, the ultimate physiological electron donor is most probably hydroquinones. Specific activities for hydroquinone oxidation are greater than or equal to those of NADH oxidation and/or protein thiol-disulfide interchange (Kishi et al., 1999, Biochim. Biophys. Acta 1412:66-77).
A constitutive CNOX form was originally defined as a drug-indifferent ECTO-NOX activity associated with the plasma membrane of non-transformed cells that was the normal cell's counterpart to tNOX (Morré, 1998, 26:421-433; DeHahn et al., 1997, Biochim. Biophys. Acta 1328:99-108; and Morré, 1998, Plasma Membrane Redox Systems and their Role in Biological Stress and Disease, Kluwer Academic Publishers, Dordrecht, N L, pp. 121-156). Indeed, a 36 kDa protein isolated from rat liver and from plants has NOX activity that is unresponsive to tNOX inhibitors (Brightman et al., 1992, Biochim. Biophys. Acta 1105:109-117).
There is a longfelt need in the art for a safe and effective nutritional supplement for treating, reducing the incidence of and/or preventing viral infections, including the common cold.